CN112480247A - Anti-human serum albumin monoclonal antibody - Google Patents
Anti-human serum albumin monoclonal antibody Download PDFInfo
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- CN112480247A CN112480247A CN202011247464.4A CN202011247464A CN112480247A CN 112480247 A CN112480247 A CN 112480247A CN 202011247464 A CN202011247464 A CN 202011247464A CN 112480247 A CN112480247 A CN 112480247A
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- serum albumin
- human serum
- monoclonal antibody
- amino acid
- hybridoma cell
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- G—PHYSICS
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Abstract
The invention relates to a human serum albumin mature peptide amino acid sequence which is analyzed by a computer, and a plurality of human serum albumin specific antigenic determinant amino acid sequence short peptides are selected by comparing the human serum albumin mature peptide amino acid sequence with the mouse, cow, monkey, sheep and rabbit down-derived albumin mature peptide amino acid sequence, wherein the length of the short peptide amino acid is 15-35 respectively. Artificially synthesized short peptide is used for immunizing mice to obtain the mouse hybridoma cell engineering strain which can generate specific anti-human serum albumin monoclonal antibody which has no immunoreaction with mice, cows, monkeys, sheep and rabbits. The antibody prepared by the cell strain has ultrahigh sensitivity and specificity to human serum albumin.
Description
Technical Field
The invention belongs to the field of biotechnology. The invention relates to a human serum albumin mature peptide amino acid sequence which is analyzed by a computer, and a plurality of human serum albumin specific antigenic determinant amino acid sequence short peptides are selected by comparing the human serum albumin mature peptide amino acid sequence with the mouse, cow, monkey, sheep and rabbit down-derived albumin mature peptide amino acid sequence, wherein the length of the short peptide amino acid is 15-35 respectively. Artificially synthesized short peptide is used for immunizing mice to obtain the mouse hybridoma cell engineering strain which can generate specific anti-human serum albumin monoclonal antibody which has no immunoreaction with mice, cows, monkeys, sheep and rabbits. The antibody prepared by the cell strain has ultrahigh sensitivity and specificity to human serum albumin.
Background
Human Serum Albumin (HSA) is a soluble monomeric protein that makes up half of the total amount of protein in the blood. Albumin, as a basic carrier, carries, inter alia, delivery fatty acids, steroids and hormone molecules, the stable inert nature of which is an important factor in maintaining blood pressure. Serum albumin is a globular, non-glycosylated, 65 kilodalton molecular weight, 585 amino acid serum protein. This protein (albumin precursor) is then secreted extracellularly by the process of conversion of the Golgi apparatus to remove the leader polypeptide. Serum Albumin has 35 cysteines, and in blood Albumin is a monomer with 17 disulfide bonds (see Brown JR, "Albumin structure, Function, and Uses" Pergamon, n.y., 1977). When the polypeptide is not secreted, the albumin product in the yeast cell is in a mismatched state, will lose 90% of its antigenicity (compared to the native state albumin in plasma), and form insoluble albumin aggregates, without the biological activity of albumin. Human serum albumin is now extracted from human plasma for clinical use.
Albumin is the main component in blood, and has a content of 40-50 g per liter of blood, and a half-life of more than 20 days. Albumin is also commonly used as a stabilizer in pharmaceutical formulations, particularly in the manufacture of biopharmaceuticals and vaccines. In conclusion, after the gene of the human serum albumin and the therapeutic protein is fused in yeast or vertebrate cells and expressed into the recombinant fusion protein by using the genetic engineering technology, the fusion protein has great advantages so that the fusion protein can resist the enzymolysis in vivo, and the service life of the therapeutic protein in vivo and in vitro can be greatly improved, namely, the stability and the longer half-life period of the therapeutic protein in serum and during storage can be increased, the long-acting effect of the protein medicament can be achieved, the administration frequency can be greatly reduced, and a better treatment effect can be obtained in the clinical treatment of major diseases (in forests, rich rocks, more excellent biological innovative medicaments, namely the recombinant human serum albumin fusion protein, Chinese medicinal biotechnology 2017, 12(3): 248-264).
When microalbumin is detected in human urine, it is reflected that the kidneys may have abnormal leakage of protein. The urine trace albumin detection index can be used as an early diagnosis index of chronic diseases such as diabetes, nephropathy and the like, and has important clinical significance for quantitatively detecting albumin in urine. Therefore, the establishment of a specific and sensitive method for detecting the microalbumin in urine also has important application value, and the key of the method lies in the sensitivity and specificity of the anti-albumin monoclonal antibody, namely the human serum albumin monoclonal antibody, on the detection sensitivity and specificity of the anti-albumin monoclonal antibody, and the clinical examination of whether a patient potentially has the symptoms of chronic diseases such as diabetes, nephropathy and the like is carried out, so that the method has important clinical significance for finding the disease condition as soon as possible and treating as soon as possible.
On the other hand, in view of the significant difficulty in blood sampling and plasma collecting of human serum albumin derived from blood sources, human albumin is a clinical treatment drug for treating various clinical indications such as shock, burn, trauma, hypoproteinemia, acute blood volume reduction, cancer chemotherapy, ascites, late cancer, and improvement of the immunity of the aged. Therefore, the production of recombinant human serum albumin (rHSA) by using microorganisms and plants, especially Pichia engineering bacteria, has become an upgrading and updating research product of HSA of blood source. The construction, production process and purification process of recombinant human serum albumin expression engineering bacteria are disclosed in China issued patents ZL2004010057313.7 and ZL2008 of forest and Rich rock. However, the purity of recombinant human serum albumin is more critical. The antibody affinity chromatography separation and purification is carried out by utilizing the characteristic that the antibody/antigen has specific recognition, the purity of the recombinant human serum albumin can be further greatly improved (the residual amount of the host protein is greatly reduced), the purification efficiency is higher, and the method has very important value on the safety and the economy of the clinical patient medication.
The anti-human serum albumin monoclonal antibodies are commercially available products, but although the antibodies have specificity, binding sites on HSA are unknown, and the antibodies are expensive, so that mouse hybridoma cell lines cannot be provided. In the preparation of antibody chromatography packing material for human serum albumin purification, especially in large-scale production, it is required that the binding site of the antibody and human serum albumin must be clear and specific. But the large-scale preparation of the antibody needs to have low cost, simplicity and reproducibility stability. This has led to the technical approach accomplished by the present invention to obtain a mouse monoclonal antibody against human serum albumin with a well-defined antibody antigen binding site.
Disclosure of Invention
The invention aims to solve the problems of large-scale production and preparation of an anti-human serum albumin monoclonal antibody, a diagnostic kit for clinically detecting trace amount of human serum albumin in a sample and an antibody affinity chromatography tool for preparing recombinant human serum albumin which can be used for production and preparation of an exogenous expression system (yeast or plant).
The invention is realized according to the following technical scheme.
The inventor utilizes computer analysis software to compare the differences of amino acid sequences of serum albumin of monkey, mouse, rabbit and sheep and human serum albumin and search epitope amino acid sequence short peptide with specificity in the amino acid sequence of human serum albumin. Artificially synthesizing the short peptides, respectively using the short peptides to immunize mice, and screening out a hybridoma cell strain which can only produce a monoclonal antibody which can only react with Human Serum Albumin (HSA) through an ascites hybridoma technology.
The preparation method of the hybridoma cell strain secreting the monoclonal antibody with specific and strong anti-human serum albumin is characterized by comprising the following steps.
1. The human serum albumin amino acid sequence which is possibly an antigenic determinant is selected by a computer, and after screening, the artificially synthesized polypeptide (short peptide) amino acid sequences are respectively:
SEQ ID No. FR1026: ENFKALVLIAFAQYLQ (16aa)
SEQ ID No. FR1027: LLRLAKTYETTLEKCCAAAD (20aa)
SEQ ID No. FR1028: CFAEEGKKLVAASQAALGL (19aa)
10mg of each short peptide was synthesized, wherein 5mg of the 10mg of short peptides were chemically coupled to KLH.
2. 200 μ g of KLH-coupled short peptide plus Freund's complete adjuvant were mixed in equal volume and emulsified thoroughly to perform basic immunization of mice by intraperitoneal injection of BalB/c mice in a volume of 0.2ml per injection, and 5 mice were immunized simultaneously with each short peptide. The immune antigen coupled KLH short peptide and Freund's incomplete adjuvant are mixed in equal volume and emulsified fully, the second immunization is performed by intraperitoneal injection for 1 time every 2 weeks, the injection volume is 0.2ml, and the dosage of the coupled KLH short peptide is 100 mu g/mouse. Mixing immunogen coupling KLH short peptide with 0.9% normal saline, 3 days before cell fusion, reinforcing the immunity of mice, injecting the mixture into the abdominal cavity, wherein the dosage of the coupling KLH short peptide is 100 mu g/mouse.
3. After the second immunization, blood can be collected from the eyeground of the mouse at proper time, and the immune effect of the mouse can be measured after serum is centrifugally taken. Collecting splenocytes from immunized mice and NS-1 myeloma cells of the immunized mice according to a conventional technology, and fusing by using polyethylene glycol (PEG) preparation according to a ratio of 6: 1; selectively culturing with HAT culture solution. 7-14 days after fusion, taking cell culture supernatant, screening hybridoma cell strains secreting anti-HSA by adopting an indirect ELISA method, and subcloning the obtained positive clone strains by adopting a limiting dilution method.
4. And repeatedly carrying out screening by using the subclone and the indirect ELISA method for several times, and obtaining at least one monoclonal antibody hybridoma cell strain secreting anti-HSA and having specificity and high sensitivity for each artificially synthesized HSA antigenic determinant short peptide.
5. A clone cell strain (FR 1028) of hybridoma obtained by short peptide SEQ ID number JN-1028 can secrete a specific and high-sensitivity anti-human serum albumin monoclonal antibody (mAb1028), and further application of the monoclonal antibody as a more excellent anti-HSA monoclonal antibody is developed.
In the present invention, a hybridoma cell line capable of producing a monoclonal antibody against human serum albumin is obtained by the following method.
1. Obtained by cell culture fluid: hybridoma cell strain FR1028 was expressed by 1 × 106The inoculum size was inoculated into 6 ml RPMI-1640 medium containing 20% fetal bovine serum at 37oC cell culture box containing 5% CO2 for 3-5 days, centrifuging the cell culture solution at 5000 Xg for 5 min, collecting supernatant, and purifying monoclonal antibody by affinity chromatography using Protein A or Protein G filler.
2. Obtained by ascites of animals: injecting BalB/c mice with 0.5 ml paraffin oil, 7-14 days later, injecting the cells obtained in step 1 into the abdominal cavity of each mouse, 1-2 × 106Ascites is extracted after the abdomen of the mouse is obviously enlarged, 4oAnd C, centrifuging at 6000 Xg for 10 minutes, collecting supernatant, and purifying the monoclonal antibody by affinity chromatography of Protein A or Protein G packing.
In the present invention, a monoclonal antibody produced by a hybridoma cell line FR1028 is characterized in that the subtype of the antibody is identified by the following method.
1. And (3) identifying the purity of the antibody: the purity of the monoclonal antibody was determined by 12% SDS-PAGE, as shown in FIG. 1. The purity of the antibody separated and enriched by the Protein-A column chromatography can be more than or equal to 95 percent.
2. And (3) antibody subtype identification: using IsoTripTMMouse monoclonal antibody typing kit (Merck company, Ltd.)Cat.no. 11493027001) and the determination result shows that the antibody subtype is IgG1 kappa type.
The invention discloses application of a hybridoma cell strain FR1028, which is characterized in that a monoclonal antibody (mAb1028) generated by the hybridoma cell strain FR1028 has ultrahigh sensitivity and ultrahigh specificity to HSA. The antigen blood source pHSA or the rHSA expressed by pichia pastoris is adopted to directly coat a 96-well plate (produced by Corning corporation), and the specific recognition capability of the monoclonal antibody (mAb1028) generated by the hybridoma FR1028 is directly measured by an ELISA method. The details are as follows.
A96-well ELISA detection plate is directly coated with HSA with different concentrations, and a blank control, a negative control and a positive control are arranged in the detection. Blocking the detection plate by 3% skimmed milk powder (Non-fat milk), and sequentially adding mAb1028 antibody, Biotin-labeled Goat anti-mouse antibody (Biotin-coat anti-mouse Ig G1), and streptavidin-labeled horseradish peroxidase (Avidin-HRP). Then adding TMB color development solution, stopping the reaction by using sulfuric acid, and finally carrying out color comparison at 450nm by using an enzyme labeling instrument. The results show (FIG. 2) that the monoclonal antibody (mAb1028) produced by the FR1028 hybridoma cells had a sensitivity and specificity for antigen recognition by HSA that reached a level of 0.0012 pg/ml (1.2 fg/ml HSA) without any reaction with Bovine Serum Albumin (BSA). It can be seen that mAb1028 recognizes HSA with ultra-high sensitivity and ultra-high specificity.
The monoclonal antibody produced by the FR1028 cells of hybridoma cells (mAb1028) has specific recognition of HSA and superior sensitivity for a variety of applications, as described below.
1. Detection of trace amounts of human serum albumin
A96-hole ELISA detection plate is respectively coated with mAb1028 or a commercial human serum albumin monoclonal antibody (cat #, produced by Sigma) in parallel, the detection plate is sealed by a commercial protein-free sealing solution, and then a sample to be detected, such as human urine (containing trace human albumin) or a human serum albumin diluent (1 pg-0.1fg/ml, diluted by 10 times and used as a standard curve of the concentration of the human serum albumin), is added. The detection is provided with a PBS blank as a negative control, and Bovine Serum Albumin (BSA) with the concentration of 200ng/ml is used as a specific control. After incubation, the cells were washed 3 times, and after adding HRP-Goat anti-mouse antibody (Biotin-Goat anti-mouse IgG 1) and TMB developing solution in this order, the reaction was stopped with 1M sulfuric acid, and finally, the cells were measured for color at 450nm using an enzyme reader.
The result shows that the monoclonal antibody (mAb1028) produced by the hybridoma FR1028 can be used for detection of an ELISA kit, and can detect that the lower limit content of HSA in diluted urine is 4.5fg/ml, and the lower limit trace of human serum albumin is 0.6 fg/ml.
1. Preparation of immunoaffinity filler for purifying human serum albumin, especially recombinant human serum albumin
The preparation method comprises the steps of using a monoclonal antibody (mAb1028, the purity is 95%) produced by a hybridoma cell strain FR1028 and a Sepharose 4B Fast Flow filler produced by activated GE as a matrix to perform chemical coupling treatment to prepare a specific human serum albumin-antibody affinity filler, filling the specific human serum albumin-antibody affinity filler into a chromatographic column, introducing a sample (fermentation supernatant or diluted human serum albumin solution) containing HSA onto the chromatographic column, removing a Flow-through liquid and a cleaning liquid, eluting human serum albumin specifically combined with the monoclonal antibody from the chromatographic filler by using an eluent, and collecting, separating and purifying the human serum albumin.
2. Is used for detecting and identifying recombinant human serum albumin fusion protein
The inventor uses mAb1028 anti-human serum albumin monoclonal antibody to test recombinant human serum albumin fusion proteins with different molecular structures and conformations, and obtains the results which are meaningful and unexpected. mAb1028 is the last 19 amino acid sequences of the C-terminus of human serum albumin. When therapeutic cytokine proteins (e.g., granulocyte stimulating factor G-CFS, interferon IFN-. alpha., growth hormone) are present at the C-terminus of human serum albumin, fusion proteins of different therapeutic cytokine proteins with human serum albumin have different immunoreactivity strengths (. mu.g to fg) for the different fusion proteins. The method shows that whether therapeutic protein is fused to the C-terminal of the human serum albumin or the binding capacity of the mAb1028 and the C-terminal amino acid sequence (antigenic determinant) of the human serum albumin is interfered, and also shows that even if the recombinant human serum albumin fusion proteins are fused to the C-terminal of the albumin, the recombinant human serum albumin fusion proteins are obviously different, and each fusion protein needs to be repeatedly and independently examined at the cellular level, the animal test level and the clinic level. When the therapeutic cytokine protein is fused with the N-terminus of human serum albumin, the differences in the immunoreaction of the mAb1028 anti-human serum albumin monoclonal antibody of the invention are not significant, for example: recombinant human interleukin-2/serum albumin fusion protein (hIL-2/SA).
In summary, the hybridoma cell strain FR1028 has the advantages that the hybridoma cell strain FR1028 obtained by the present invention can specifically secrete a monoclonal antibody against HSA, and has ultrahigh sensitivity and ultrahigh specificity to HSA. Compared with the existing commercial monoclonal antibody product of anti-human serum albumin, the specific monoclonal antibody has wide practical application in detection of ultra-trace albumin, preparation of human urinary albumin detection kit, purification of recombinant human serum albumin and the like.
The invention also has the advantages that the monoclonal antibody produced by the cell strain obtained by the invention has multiple purposes and has very good value in actual production and clinical application.
Drawings
FIG. 1: examples of mouse specific HSA monoclonal antibodies generated from hybridoma cell lines. MK represents a molecular weight protein standard; A. b, C is mAb1026, mAb1027 and mAb1028 murine monoclonal antibodies (added reducing agent DTT, heated to boiling); D. e, F the rat monoclonal antibody was added to the sample after boiling for 10 minutes without adding DTT as a reducing agent. In the reduced state (A, B, C) the murine single antibody can then be split into two fragments (50 kD and 23 kD). In the non-reduced state, the murine monoclonal antibody appears as a band with a molecular weight of about 150 kD.
FIG. 2: ELISA tests the sensitivity and specificity of the monoclonal antibody produced by hybridoma FR1028 on antigen recombinant human serum albumin and short peptide SEQ ID No. 1028. In the figure, the abscissa represents the concentration of recombinant human serum albumin and the concentration of short peptide 1028 as antigen, and the ordinate represents OD450And (6) reading.
Detailed Description
Example 1 computer software analysis for the confirmation of antigenic determinants and the Synthesis of antigenic determinant Polypeptides
Human, bovine, monkey, murine, ovine and rabbit serum albumin amino acid sequences were subjected to molecular structural analysis to confirm possible epitope amino acid polypeptide sequences. These polypeptides (short peptides) were manually selected and then subjected to synthesis.
The human serum albumin selected by the artificial synthesis computer can be amino acid sequence polypeptide-short peptide of an antigenic determinant, and then the following amino acid sequence polypeptide is selected and synthesized by confirmation to carry out the preparation research of the anti-human serum albumin monoclonal antibody. The artificially synthesized polypeptide amino acid sequences are respectively as follows:
SEQ ID No. FR-1011：VRPEVDVMCTAFHDNEET (18aa，140-157aa)
SEQ ID No. FR-1012：LLRLAKTYETTLEKCCAAAD (20aa，370-389aa)
SEQ ID No. FR-1013：AKVFDEFKPLVE (12aa，395-406aa)
SEQ ID No. FR-1014：SKCCKHPEAKRM (12aa，459-470aa)
SEQ ID No. FR-1015：VPKEFNAETFTF (12aa，522-533aa)
SEQ ID No. FR-1026：ENFKALVLIAFAQYLQ (16aa，41-61aa)
SEQ ID No. FR-1027：LLRLAKTYETTLEKCCAAAD (20aa，370-389aa)
SEQ ID No. FR-1028：CFAEEGKKLVAASQAALGL (19aa，591-609aa)
polypeptides that are considered to be determinants of human serum albumin are approximately 12-20 amino acids in length. The inventor selects three polypeptides of SEQ ID number FR-1026, SEQ ID number FR-1027 and SEQ ID No. FR-1028 to be artificially synthesized, each short peptide is synthesized into 10mg, 5mg of the 10mg short peptides are chemically coupled with KLH, and the short peptides are directly used for immunizing mice together with an adjuvant.
Example 2 immunization of mice and establishment of hybridoma cell lines
200 μ g of KLH-conjugated short peptide plus Freund's complete adjuvant were mixed in equal volume and emulsified thoroughly to perform basic immunization of BalB/c mice by intraperitoneal injection of BalB/c mice in a volume of 0.2ml per injection, and 5 mice were immunized simultaneously with each short peptide. The booster immunization is prepared by mixing 100 μ g of immune antigen coupled with KLH short peptide and Freund's incomplete adjuvant in equal volume, emulsifying thoroughly, and performing intraperitoneal injection 1 time every 2 weeks, wherein the injection volume is 0.2 ml. 3 days before cell fusion, mice were boosted with 100. mu.g of KLH-conjugated short peptide and mixed with 0.89% physiological saline, and injected intraperitoneally. The second immunization is started, and the eyeground blood of the mouse is taken each time to carry out immunoreaction measurement to determine whether to continue the immunization, and the immunization is generally repeated for 4 times at most.
Example 3 preparation of mouse hybridoma cells
Preparation of NS-1 myeloma cells: NS-1 cells with good growth status were selected, washed once with culture medium, and NS-1 myeloma cells were gently aspirated down with 10 ml of culture medium.
Preparation of mouse splenocytes: mice 3 days after the last 1 booster immunization were taken, and the eyeballs were removed to collect blood for positive serum isolation. Killing the mouse by using a cervical dislocation method, opening the abdominal cavity of the mouse by a sterile method, taking out the spleen, washing the spleen by using a DMEM culture solution, and carefully removing connective tissues attached to the spleen. The spleen was then transferred to another dish containing DMEM medium. Squeezing with forceps to release splenocytes from mouse, and making splenocyte suspension.
Cell fusion: NS-1 myeloma cells prepared as described above were mixed with spleen cells, centrifuged at 800 Xg for 5 minutes, and the supernatant was discarded. The bottom of the cell tube was gently shaken to mix the two cells well. The preheated PEG was slowly added to the fusion tube over 1 minute with a pipette while gently shaking and mixing. Then adding human 37oC preheated DMEM, supplementing 5 ml of HAT culture medium, gently suspending and precipitating cells, then adding a proper amount of freshly prepared mouse ascites containing macrophages, and finally supplementing HAT culture medium to 50 ml. The cells were distributed in 96-well cell culture plates, each 100. mu.l, and the 96-well cell culture plates were then cultured in 37 ℃ 5% CO2 cell culture chambers. Half of the medium was changed with HAT medium after 5 days. Observing the growth condition of the hybridoma cells every day, sucking a proper amount of cell supernatant when the cell culture supernatant turns yellow or clones to be distributed to more than 1/10 of the area of the bottom of a hole, screening hybridoma cell strains secreting anti-human serum albumin antibodies by adopting an indirect ELISA method, and subcloning the obtained positive clone strains by adopting a limiting dilution method.
Screening hybridoma cell strains: through 3-6 times of subcloning and indirect ELISA screening, hybridoma cell strains with a plurality of cell strains capable of stably secreting monoclonal antibodies are screened. The obtained immune hybridoma cell strains which stably secrete the monoclonal antibodies are respectively named and preserved for seed production for further analysis and determination.
Example 4 method for measuring the titer of monoclonal antibodies produced by mouse hybridoma cell lines
Taking a mouse hybridoma cell strain FR1028 as an example, the cell strain is cultured in RPMI-1640 medium containing 10-20% fetal bovine serum for passage, and the cell strain is subjected to passage once every 3 days, and a sample is reserved for each passage to be subjected to monoclonal antibody determination.
Example 5 measurement of cell culture supernatant titer
Passaged cells were arranged at 1X 106The cell number was inoculated into 6 ml of RPMI-1640 medium containing 20% fetal bovine serum at 37oC cell culture box containing 5% CO2 for 3-5 days, centrifuging the cell culture solution at 800 Xg for 5 min, collecting supernatant, and measuring monoclonal antibody titer in the cell supernatant by indirect ELISA method.
Example 6 measurement of ascites titer in mice
BalB/c mice were intraperitoneally injected with 0.5 ml of paraffin oil, and 7-14 days later, each mouse was intraperitoneally injected with 1X 10 cells obtained in example 36Ascites is extracted after the abdomen of the mouse is obviously enlarged, 4oAt C, 8000 Xg for 10min, and collecting the supernatant. The monoclonal antibody in the supernatant is measured by an indirect ELISA method, and the titer is 1:1 × 105-1:5×105。
EXAMPLE 7 subculture of hybridoma cell lines
Culturing and passaging the cell strain FR1028 in an RPMI-1640 culture medium containing 20% fetal calf serum once every 3 days until the cell strain FR1028 is passaged to 30 generations, wherein the hybridoma cell strain FR1028 can still grow well and stably passaged, and the titer of a culture solution supernatant can still reach the level of 1:50,000-1:100,000. Therefore, the cell strain FR1028 can be stably passaged, and can continuously and stably produce the monoclonal antibody of the anti-human serum albumin.
EXAMPLE 8 preparation of monoclonal antibody against human serum albumin Using cell line FR1028
The monoclonal antibody is prepared by injecting BalB/c mouse with 0.5 ml paraffin oil, injecting 1-2 × 106 hybridoma FR1028 cells into the abdominal cavity of each mouse 7-14 days later, extracting ascites after the abdominal cavity of the mouse is obviously enlarged, centrifuging at 8000 × g at 4 ℃ for 10min, and collecting supernatant. Then, the supernatant of the abdominal water was purified by using Protein A-Sepharose 4 Fast Flow packing (manufactured by GE Healthcare). The flow-through and wash-off were discarded and then eluted with glycine-HCl, pH 3.0. The eluate was collected and neutralized to pH6.5 with Tris-HCl, 1M, pH9.0 buffer to obtain anti-human serum albumin monoclonal antibody (mAb 1028).
Example 9 identification of monoclonal antibody mAb1028
And (3) identifying the purity of the antibody: the purity of the murine mAb can be identified by SDS-PAGE electrophoresis using a 12.5% separation gel. FIG. 1 shows an electrophoresis diagram of an example of mouse-specific HSA mab produced from hybridoma cell lines. MK represents a molecular weight protein standard; A. b, C is mAb1026, mAb1027 and mAb1028 murine monoclonal antibodies (added reducing agent DTT, heated to boiling); D. e, F the rat monoclonal antibody was added to the sample after boiling for 10 minutes without adding DTT as a reducing agent. In the reduced state (A, B, C) the murine single antibody can then be split into two fragments (50 kD and 23 kD). In the non-reduced state, the murine monoclonal antibody appears as a band with a molecular weight of about 150 kD.
The purity of the murine monoclonal antibody mAb1028 purified by Protein A filler can reach more than or equal to 95 percent.
And (3) antibody subtype identification: the subtype of the antibody was identified using a mouse antibody typing kit (manufactured by Sigma Co.), and the specific operation was carried out according to the instructions provided by the manufacturer. The determination result shows that the antibody subtype is IgG1 kappa type.
Sensitivity identification of antibody and antigen: a96-well plate (produced by Corning corporation) is coated by recombinant human serum albumin or polypeptide SEQ ID number FR1028 with different concentrations, and the detection sensitivity and the sensitivity of the monoclonal antibody mAb1028 are measured by an ELISA direct method. The detection results are shown in table 1:
the graph of the immunity sensitivity detection is shown in figure 2: ELISA tests the sensitivity and specificity of the monoclonal antibody produced by hybridoma FR1028 on antigen recombinant human serum albumin and short peptide SEQ ID No. 1028. In the figure, the abscissa represents the concentration of recombinant human serum albumin and the concentration of short peptide 1028 as antigen, and the ordinate represents OD450And (6) reading.
The last 3 dilutions of Table 1 were selected and continued to serially dilute 4 dilutions by 3-fold dilution to 11.29, 3.76, and 1.25fg/ml, respectively. The PBS and the sera of cattle, rabbit, monkey and mouse have no immunoreaction, and the determination results in Table 2 show that the mAb1028 monoclonal antibody also has the required specificity.
Identification of specificity of antigen and antibody: mAb1028 was significantly immunoreactive with bovine serum, rabbit serum, monkey serum and mouse serum. Therefore, mAb1028 has specific immunoreaction with human serum albumin only, can identify human serum albumin, and has ultrahigh sensitivity and ultrahigh specificity.
Example 10 detection of various recombinant human serum Albumin fusion proteins by mAb1028 monoclonal antibodies
The recombinant human serum albumin fusion protein (table 2) can be obtained by directly gene-fusing therapeutic cytokine protein, and the molecular structure can be obtained by directly fusing therapeutic protein mature peptide and the N-terminal or C-terminal of human serum albumin mature peptide without connecting peptide therebetween. The yeast or CHO cell-expressed production preparation is then selected depending on whether the therapeutic protein is non-glycosylated (yeast is used) or glycosylated (CHO cells are required). Theoretically, direct attachment of the therapeutic protein to the C-terminus of human serum albumin could affect the surface antigen binding domain (epitope) recognized by the mAb1028 monoclonal antibody. Because mAb1028 was a monoclonal antibody prepared using the last 19 amino acid sequences of the C-terminus of human serum albumin.
The method is that recombinant human serum albumin fusion protein (100 mu l/hole) with different molecular structures is coated in a gradient way, and PBS 4 is diluted for overnight at 4 ℃. The coating solution was poured off, the plate washed 3 times, and blocking solution, 1% BSA, 200. mu.l/well, 37 ℃ for 2h was added. Pour off, pat dry, add mAb1028 murine mAb diluted 50000 fold, 100. mu.l/well, 37 degrees, 1 h. The incubation was decanted, PBST 300. mu.l/well, plate washed 5 times, and patted dry. HRP-labeled Goat Anti-Mouse secondary antibody was added, and PBST was diluted 1: 5000-fold, 100. mu.l/well, 37 ℃ and incubated for 1 h. Pour off, PBST 300. mu.l/well, wash plate 5 times, pat dry. Then adding TMB 100 mul/hole, 37 degree, 10min, adding 1M sulfuric acid to stop reaction, measuring OD value at 450 nm. The results of the analytical experiments are shown in Table 3.
The results show that the fusion protein formed by directly connecting different therapeutic cytokines with the C terminal of the human serum albumin has certain sensitivity difference due to different therapeutic proteins; the therapeutic cytokine protein was directly linked to the N-terminus of human serum albumin, and theoretically did not significantly affect the binding ability of the monoclonal antibody of the invention that recognizes only the C-terminus of human serum albumin (code: 9555 is human interleukin-2 variant fusion protein at the N-terminus of the mature peptide of human serum albumin, rhIL-2/SA). Meanwhile, the sensitivity of different fusion proteins expressed by the yeast can have immunoreaction difference which is nearly 1 time or more. No particular differences appear to have been shown between the use of different expression production systems, either Pichia or CHO cells. This is because 1882(rHSA/EPO) is expressed by CHO cells, EPO is at the C-terminal end of human serum albumin and is directly linked, which is the same as 9075(rHSA/GH) expressed by yeast engineering bacteria, GH is at the C-terminal end of human serum albumin and is directly linked, and there is no significant difference in the immunoreaction sensitivity of the two antibodies on the same antibody.
Meanwhile, the experiment investigates that the fusion protein still has more obvious steric hindrance effect compared with the human serum albumin monomer. That is, if 1 fusion protein binds to 1 antibody when immunoaffinity reacting with an ELISA plate coated with a monoclonal antibody, the larger fusion protein occupies more antibody space around the antibody, so that it cannot bind to the antigen any more, and as a result, the detection sensitivity is greatly decreased. This is not less than a problem with the sensitivity and specificity of the immunoreaction, but rather is due to steric hindrance.
EXAMPLE 10 monoclonal antibody produced by cell line FR1028 for preparing human serum albumin assay kit
The mAb1028 monoclonal antibody produced based on the immune hybridoma cell strain FR1028 has ultrahigh sensitivity and ultrahigh specificity to antigen human serum albumin, and can be applied to detection of trace amounts or trace amounts of residual human serum albumin in samples. The specific method is described below. By adopting an ELISA antibody sandwich method, the primary antibody is mAb1028, and the secondary antibody is commercial American Sigma Cat # A6684 anti-human serum albumin monoclonal antibody or rabbit anti-human serum albumin polyclonal antibody. Horseradish peroxidase-labeled HPR-goat anti-mouse or rabbit monoclonal antibodies were used as the enhancing chromogenic agents. The blocking solution used was a protein-free blocking solution (Shanghai). The sample to be tested was added and the volunteer urine was diluted with 0.9% physiological saline to obtain a 100. mu.l sample. Meanwhile, human serum albumin with different concentrations is used for making a protein standard curve, which is 1ng/ml, 100pg/ml, 10pg/ml, 1pg/ml, 100fg/ml, 10fg/ml, 1fg/ml, 0.1fg/ml and 0.01fg/ml respectively. Detection of blank control: PBS was used instead of HSA; negative control: bovine Serum Albumin (BSA) was used at a concentration of 50 mg/ml; adding 100 μ l of single-component TMB for color development, stopping the reaction with 1N sulfuric acid, and finally performing color comparison at 450nm in a microplate reader. Duplicate wells were set up for the experiments, with 2 duplicates per sample. By OD450The reading is determined for the test result. The results show that trace albumin in urine of volunteersThe content is 1-10 fg/ml.
EXAMPLE 11 monoclonal antibody produced by cell line FR1028 for use in immunoaffinity chromatography
The filler for column chromatography of affinity antibody for separation and purification of human serum albumin is NHS-activated Sepharose 4 Fast Flow available from GE Healthcare, Cat #17090601, 17090602 or 17090604. NHS (N-hydroxysuccinimide) coupling with ligands containing primary amine groups can form chemically stable amide bonds. The mAb1028 antibody was coupled to the activated sepharose packing as provided by the manufacturer using the instructions. Blocking the active groups which are not coupled by using a blocking solution, and treating by using a washing buffer solution to obtain the filler for the human serum albumin affinity chromatography purification purpose. The results show that the binding capacity of the mg antibody and the activated agarose gel filler per liter L is about 20-30mg/L, which meets the technical index of manufacturers.
mAb1028 immunoaffinity chromatography column prepared in example 12 for purification of human serum albumin
The immunoaffinity chromatography filler coupled with the monoclonal antibody is filled in a human chromatographic column, and after being balanced by balance buffer solution (20 mM Na2HPO4 and NaH2PO4, pH7.2), a crude pure intermediate product of recombinant human serum albumin separated and enriched by Capto-MMC is introduced. The method for detecting the residual amount of host protein/HCP of the pichia pastoris can be seen in the invention patent application publication number of the inventor: CN 109851674A. The flow-through was left as staged for analysis of human serum albumin loading. Balancing by balance liquid after sample loading; 20mM Na was used2HPO4And NaH2PO4, 2M NaCl, pH7.2 as eluent. The results indicate that the affinity loading of albumin can be approximately between 40-50mg HSA/ml.
The results show that the purity of rHSA produced by expressing Pichia pastoris separated and enriched by Capto-MMC is about 75% before sample loading, the residual amount of host protein is about 50ng/mg, the purity of rHSAHPLC purified by mAb1028 immunoaffinity chromatography column is 99%, and the residual amount of host protein (HCP) is not detected.
Therefore, the monoclonal antibody (mAb1028) produced by hybridoma FR1028 obtained by immunizing mice can be used for preparing immunoaffinity filler, and is remarkably characterized in that when the monoclonal antibody is used for separating and enriching human serum albumin or recombinant human serum albumin in culture solution, compared with the separation and purification process of a recombinant human serum albumin and fusion protein thereof invented by the inventor in the prior art, the patent number is as follows: ZL200810089645.1, can obtain recombinant human serum albumin with higher purity and better yield. The inventor confirms that the mouse monoclonal antibody has extremely high commercial and industrial application prospects.
Sequence listing
<120> anti-human serum albumin monoclonal antibody
<141> 2020-11-09
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> SEQ ID No. FR1026
<400> 1
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
1 5 10 15
<210> 2
<211> 20
<212> PRT
<213> SEQ ID No. FR1027
<400> 2
Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys
1 5 10 15
Ala Ala Ala Asp
20
<210> 3
<211> 19
<212> PRT
<213> SEQ ID No. FR1028
<400> 3
Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala
1 5 10 15
Leu Gly Leu
<210> 4
<211> 18
<212> PRT
<213> SEQ ID No. FR1011
<400> 4
Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu
1 5 10 15
Glu Thr
<210> 5
<211> 20
<212> PRT
<213> SEQ ID No. FR1012
<400> 5
Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys
1 5 10 15
Ala Ala Ala Asp
20
<210> 6
<211> 12
<212> PRT
<213> SEQ ID No. FR1013
<400> 6
Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu
1 5 10
<210> 7
<211> 12
<212> PRT
<213> SEQ ID No. FR1014
<400> 7
Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
1 5 10
<210> 8
<211> 12
<212> PRT
<213> SEQ ID No. FR1015
<400> 8
Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
1 5 10
Sequence listing
<110> Zhongmeifuyuan Biotechnology (Beijing) Ltd
TIANJIN SINOBIOTECH Ltd.
BEIJING MEIFUYUAN BIOMEDICAL TECHNOLOGY Co.,Ltd.
TIANJIN LINDA BIOTECHNOLOGY Co.,Ltd.
<120> anti-human serum albumin monoclonal antibody
<141> 2020-11-09
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> PRT
<213> SEQ ID No. FR1026
<400> 1
Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
1 5 10 15
<210> 2
<211> 20
<212> PRT
<213> SEQ ID No. FR1027
<400> 2
Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys
1 5 10 15
Ala Ala Ala Asp
20
<210> 3
<211> 19
<212> PRT
<213> SEQ ID No. FR1028
<400> 3
Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala
1 5 10 15
Leu Gly Leu
<210> 4
<211> 18
<212> PRT
<213> SEQ ID No. FR1011
<400> 4
Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu
1 5 10 15
Glu Thr
<210> 5
<211> 20
<212> PRT
<213> SEQ ID No. FR1012
<400> 5
Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys
1 5 10 15
Ala Ala Ala Asp
20
<210> 6
<211> 12
<212> PRT
<213> SEQ ID No. FR1013
<400> 6
Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu
1 5 10
<210> 7
<211> 12
<212> PRT
<213> SEQ ID No. FR1014
<400> 7
Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
1 5 10
<210> 8
<211> 12
<212> PRT
<213> SEQ ID No. FR1015
<400> 8
Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
1 5 10
Claims (9)
1. The amino acid sequence and spatial conformation of the human serum albumin are analyzed by a computer, an amino acid sequence area which possibly has a specific surface antigenic determinant is selected, and the selected amino acid sequence short peptide is artificially synthesized and used for immunizing a mouse to prepare a mouse immune hybridoma cell which can secrete a monoclonal antibody for resisting the human serum albumin.
2. The monoclonal antibody of claim 1, derived from a human serum albumin surface epitope, the amino acid sequence of the selected epitope being SEQ ID number FR1026, SEQ ID number FR1027, and SEQ ID number FR 1028.
3. The artificially synthesized oligopeptide according to claim 1, wherein the monoclonal antibody is an IgG1 subtype antibody, and is capable of generating an antigen-specific reaction with human serum albumin by constructing an antibody expression mouse hybridoma cell line and generating the monoclonal antibody against human serum albumin after immunizing a mouse.
4. The murine anti-human serum albumin monoclonal antibody according to claim 1, preferably mAb1026, mAb1027 and mAb1028, more preferably mAb 1028.
5. The monoclonal antibody produced by the murine hybridoma cell line of claim 1, which can be used clinically for detection of trace amounts of human serum albumin.
6. The monoclonal antibody produced by the murine hybridoma cell line according to claim 1, wherein the monoclonal antibody can be used for preparing an ELISA detection kit, and the kit can be used for the detection purpose requirement of trace amount of human serum albumin.
7. The monoclonal antibody produced by the murine hybridoma cell line of claim 1, wherein the monoclonal antibody produced by the murine hybridoma cell line is used for purifying human serum albumin derived from blood or for purifying recombinant human serum albumin produced by an exogenous expression system.
8. Use according to claim 7, characterized in that monoclonal antibodies produced by the murine hybridoma cell line are used for the preparation of specific immunoaffinity chromatography packings useful for the purification of recombinant human serum albumin.
9. The monoclonal antibody produced by the murine hybridoma cell line of claim 1, which can be used in industrial production.
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| US20160251638A1 (en) * | 2013-10-31 | 2016-09-01 | Resolve Therapeutics, Llc | Therapeutic nuclease-albumin fusions and methods |
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| JP2003252899A (en) * | 2002-03-04 | 2003-09-10 | Nippon Meat Packers Inc | Epitope having immunologic reactivity for farm animal serum albumin |
| US20160251638A1 (en) * | 2013-10-31 | 2016-09-01 | Resolve Therapeutics, Llc | Therapeutic nuclease-albumin fusions and methods |
| CN104130978A (en) * | 2014-05-29 | 2014-11-05 | 齐智 | Hybridoma cell strain 11A2 and application thereof |
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| JEANNE MADISON等: "《Genetic variants of human serum albumin in Italy: Point mutants and a carboxyl-terminal variant》", 《PROC. NATI. ACAD. SCI. USA》, vol. 91, 31 July 1994 (1994-07-31), pages 2 * |
| LAJLA BRUNTSE HANSEN 等: "Identification and mapping of linear antibody epitopes in human serum albumin using high-density Peptide arrays", PLOS ONE ., vol. 8, no. 7, XP055452753, DOI: 10.1371/journal.pone.0068902 * |
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